Friday 10/26/12

10:20 A – 7:30PM

Fly Work
  •  Set up collection cages for TRiP knockdown experiments:
    • MTD x Psc 1
    • MTD x Pc 3
  • Set up new collection cage of MTD controls.  Pc labels looked weak and Pc in situs did not work at all. Try again with shorter prefix (15 min), shorter post fix (5 min?) and longer time in methanol to remove membranes (1 week?).  I bet it’s methanol, yw done in parallel were 2-3 months in MeOH at -20C.
  • All cages laying very well.  Should fix?
Cell culture prep
  • Previous cell culture split too dilute — cells not growing well.  Split plate again at 50:50.
  • added cells to 8 well chambered slides and 2 60mm petri-dishes containing coated coverslips.
  • probes to test:
    • Mes2 = very ON PolII (Red)
    • Neu3 = okay ON PolII (yellow/black/red in Kc).  Inside 120kb mostly very active transcription region.
    • Rho = okay (stalled and ON?) yellow
    • cact = okay (stalled and ON?) red
    • neur = okay (stalled and ON?)  red/yellow/blue
    • Notch = okay (stalled and ON?) red/yellow/blue
    • tkv = okay Pol II, some good spots (Pc/red)
  • Methanol treat?
  • Protocols.  methanol or ethanol stored fixed cells:  protocol1,
  • protocol2: triethylamine, acetic anhydride, and Hcl permiabilize
  • other protocols
  • Does draq5 photoswitch?
DNA FISH probe building
  • check plates — all bustling with colonies.  Moved to 4C, pick colonies tonight
  • Attempt calibration of nicked translation reactions again with (using single tube of Toll (8ul at 250ng/uL, using original Nick-Mix)
  • Looks like good smear.
  • Gel test red label BAC DNA (using 488 illumination. looks good in all channels)
  • Attempting synthesis with
    •  8 uL of red label BAC 1: at 4:1 and
    • 1:1 Enzyme mix: just PolI mix
    • 5 uL of magenta label BAC 1 at 1:1 Enzyme: just PolI mix
    • all with Cy5 dNTPs (now at .2 mM stock solution instead of 1 mM).
  • Select regions
    • GREEN (centromeric probe).  Chr2R 400,000:1,500,000
    • BLUE (en locus.  BX-C locus?)
    • RED/YELLOW (Neu3 locus),  Inos:PhR (~500kb on 2R)
    • BLACK  chr2R: 15,655,000 – 16,100,000.  sweet spot: 15,725,000 – 15,875,000.  has 2 ~10-20 kb satellite regions may need to exclude.
  • Pick colonies: (y, toll, sxl, Antp, AbdA, Ubx), growing in new LB amp O/N (7PM-)
Writing BJ Review

  • Talk to Graham about STORM II timeline (need 3D imaging for cell-culture).
    •  Hope to be online early next week.
    • be ready for software install early next week.
  • Labeling Report: Pc not good, yws look good.
  • Confocal: dl/twi double hets O/N (20 positions).
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